1. Field of the Invention
This invention relates to a method to detect methylthioadenosine phosphorylase deficiency in mammalian cells, a condition which is indicative of malignancy in those cells. Detection of cells which are deficient in this enzyme allows those cells to be targeted in chemotherapy to exploit the inability of the cells to convert methylthioadenosine to methionine.
2. History of the Invention
The amino acid methionine (MET) is necessary for the growth of normal and malignant cells. In certain malignant cells this requirement is absolute, i.e., without an adequate supply of MET, the cells die.
In mammalian cells, MET is obtained from three sources. It can be obtained in the diet, or through biochemical synthesis of MET from L-homocysteine (homocysteine) or methylthioadenosine (MTA) (a product of the polyamine biosynthetic pathway). In the latter case, MTA is converted to MET by methylthioadenosine phosphorylase (MTAse; EC 2.4.2.28).
In the past decade, researchers have identified many malignant cell lines which lack MTAse and cannot, therefore, convert MTA to MET. For example, Katamari, et al., Proc. Nat'l Acad. Sci. USA, 78: 1219-1223 (1981) reported that 23% of 3 human malignant tumor cell lines lacked detectable MTAse, while MTAse activity was present in each of 16 non-malignant cell lines studied. MTAse deficiency has also been reported as a characteristic of non-small cell lung cancers (see, Nobori, et al., Cancer Res. 53:1098-1101 (1991)), in 6 lines of lymphoma and leukemia cells (id.), in brain tumor cell lines and primary brain tumor tissue samples (id.), and in other malignancies (see e.g., Kries, et al., Cancer Res. 33:1866-1869 (1973), Kries, et al., Cancer Trmt. Rpts. 63:1069-1072 (1979), and Rangione, et al., Biochem. J 281:533-538 (1992)). MTAse negative cells principally fulfill their requirement for MET through conversion of homocysteine. However, when homocysteine is not available, the cells will generally die.
L-methionine-L-deamino-y-mercaptomethane lyase (ED 4.4.1.11; METase) is known to degrade not only MET but also homocysteine. Theoretically, therefore, one could starve malignant cells which lack MTAse (i.e., MTAse negative cells) by degrading plasma MET and homocysteine with METase. Normal MTAse positive cells would be expected to fulfill their requirement for MET by the continued conversion of MTA to MET.
One obstacle to the development of a successful approach to MET starvation of malignant cells has been the need to identify which malignancies are suitable targets for the therapy; i.e., which malignancies are MTAse negative. To that end, an assay was developed which predicts whether a malignancy is MTAse negative by determining whether any catalytic activity is present is a cell culture (Seidenfeld, et al., Biochem. Biophys. Res. Commun., 95:1861-1866, 1980). However, because of the commercial unavailability of the radiochemical substrate required for the assay, its use in routine evaluations is not presently feasible. Moreover, the assay does not account for the catalytic lability of MTAse in vitro by detecting whether any of the enzyme is present in the cell culture regardless of whether it is catalytically active at the time that the assay is performed.
This limitation of the activity assay could be avoided by the development of an immunoassay which is sufficiently sensitive to detect relatively minute quantities of enzyme. However, the purification of the MTAse enzyme from natural sources to develop antibodies for use in immunological detection of MTAse has proven to be a laborious process which produces relatively poor yields (Rangione, et al., J. Biol. Chem , 261:12324-12329, 1986).
The lack of a simple, efficient means of identifying MTAse deficient cells has contributed in part to the continued unavailability of an effective therapeutic approach to selective in vivo MET starvation of MTAse deficient malignant cells. The present invention addresses this need by providing a method for detection of the presence or absence in a sample of the gene which encodes for MTAse and by providing a recombinant source of MTAse.